The objective of this research project is to establish the identity, mechanism of action, and importance of the cellular regulator of adenylate cyclase obtained from the cytosol of rat reticulocytes. The reticulocyte cytosol activator protein (RCAP), that augments catecholamine-sensitive adenylate cyclase in rat reticulocyte membranes, has been only partially purified and characterized. The goals of the research plan are to purify and to completely characterize RCAP; to elucidate the mechanisms by which it so profoundly influences catecholamine responsiveness; and to establish to what extent, and by what means, RCAP regulates adenylate cyclase activity in other tissues and species. RCAP will be purified to homogeneity using established biochemical techniques including preparative isoelectric focusing and other specific approaches outlined in Methods. The mechanism of action of RCAP will be investigated by studies directed at RCAP's effects upon each of the three components of the hormonal unit, the beta-adrenergic receptor, the guanine nucleotide coupling protein, and the catalytic unit of adenylate cyclase, as well as the interactions among them. Specific methods will include radioligand binding studies, determination of adenylate cyclase activity, and assessment of the guanine nucleotide binding protein by means of the ADP-ribosylation assay. The effects of RCAP upon other hormones that stimulate or inhibit adenylate cyclase and in several other mammalian and tissues will also be investigated in order to determine the importance of RCAP as a general regulator of hormone action.